5-Aminolevulinic Acid Protects Against Cisplatin-Induced Nephrotoxicity Without Compromising the Anticancer Efficiency of Cisplatin in Rats in Vitro and in Vivo

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5-Aminolevulinic Acid Protects against Cisplatin-Induced Nephrotoxicity without Compromising the Anticancer Efficiency of Cisplatin in Rats In Vitro and In Vivo

Yoshio Terada1*., Keiji Inoue2., Tatsuki Matsumoto1, Masayuki Ishihara1, Kazu Hamada1, Yoshiko Shimamura1, Koji Ogata1, Kosuke Inoue1, Yoshinori Taniguchi1, Taro Horino1, Takashi Karashima2, Kenji Tamura2, Hideo Fukuhara2, Shimpei Fujimoto1, Masayuki Tsuda3, Taro Shuin2 1 Department of Endocrinology, Metabolism and Nephrology, Kochi Medical School, Kochi University, Kohasu, Oko-cho, Nankoku, Japan, 2 Department of Urology, Kochi Medical School, Kochi University, Kohasu, Oko-cho, Nankoku, Japan, 3 Institute for Laboratory Animal Research, Kochi Medical School, Kochi University, Kohasu, Oko-cho, Nankoku, Japan

Abstract

Background/Aims: Nephrotoxicity is a frequent and major limitation in cisplatin (CDDP)-based chemotherapy. 5- Aminolevulinic acid (ALA) is widely distributed in animal cells, and it is a precursor of tetrapyrole compounds such as heme that is fundamentally important in aerobic energy metabolism. The aim of this study is to evaluate the protective role of ALA in CDDP-induced acute kidney injury (AKI).

Method: We used CDDP-induced AKI rat model and cultured renal tubular cells (NRK-52E). We divided four groups of rats: control, CDDP only, CDDP + ALA(post);(ALA 10 mg/kg + Fe in drinking water) after CDDP, CDDP + ALA(pre & post).

Result: CDDP increased Cr up to 6.5 mg/dl, BUN up to 230 mg/dl, and ALA significantly reduced these changes. ALA ameliorates CDDP-induced morphological renal damages, and reduced tubular apoptosis evaluated by TUNEL staining and cleaved caspase 3. Protein and mRNA levels of ATP5a, complex(COX) IV, UCP2, PGC-1a in renal tissue were significantly decreased by CDDP, and ALA ameliorates reduction of these enzymes. In contrast, Heme Oxigenase (HO)-1 level is induced by CDDP treatment, and ALA treatment further up-regulates HO-1 levels. In NRK-52E cells, the CDDP-induced reduction of protein and mRNA levels of mitochondrial enzymes was significantly recovered by ALA + Fe. CDDP-induced apoptosis were ameliorated by ALA + Fe treatment. Furthermore, we evaluated the size of transplantated bladder carcinoma to the rat skin, and ALA did not change the anti cancer effects of CDDP.

Conclusion: These data suggested that the protective role of ALA in cisplatin-induced AKI is via protection of mitochondrial viability and prevents tubular apoptosis. Also there are no significant effects of ALA on anticancer efficiency of CDDP in rats. Thus, ALA has the potential to prevent CDDP nephrotoxicity without compromising its anticancer efficacy.

Citation: Terada Y, Inoue K, Matsumoto T, Ishihara M, Hamada K, et al. (2013) 5-Aminolevulinic Acid Protects against Cisplatin-Induced Nephrotoxicity without Compromising the Anticancer Efficiency of Cisplatin in Rats In Vitro and In Vivo. PLoS ONE 8(12): e80850. doi:10.1371/journal.pone.0080850 Editor: Jeff M. Sands, Emory University, United States of America Received August 7, 2013; Accepted October 13, 2013; Published December 6, 2013 Copyright: ß 2013 Terada et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by The Kidney Foundation Japan and the Kochi organization for medical reformation and renewal (to YT); grants from the Ministry of Education, Science, Culture and Sports of Japan (to YT, KI, YS, KI, SF, TS); and a grant-in-aid for Progressive Renal Diseases Research, Research on Rare and Intractable Disease, from the Ministry of Health, Labour and Welfare of Japan (to YT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work.

Introduction caspase-3 [4]. Oxidant stress also appears to contribute to the cisplatin-induced apoptosis of renal tubular cells, both in vitro and Cisplatin is one of the most effective and potent anticancer in vivo [5]. Several studies, including ours, suggest that caspase drugs in the treatment of epithelial malignancies such as lung, inhibitors and knockout of apoptosis-related genes attenuate head and neck, ovarian, bladder, and testicular cancers [1]. The cisplatin-induced acute kidney injury (AKI) in rats [6,7]. Mito- major constraint to cisplatin-based chemotherapy is the frequent chondria have a variety of important intracellular functions, development of nephrotoxicity [2]. The antineoplastic effect of including ATP production, synthesis of reactive oxygen species, cisplatin is dose dependent, yet the risk of nephrotoxicity often and regulation of the cell death pathway. Recent studies, including precludes the use of higher doses to maximize the therapeutic ours, have demonstrated that mitochondrial function is one of the effect. Cisplatin induces apoptosis of renal proximal tubule cells key factors protecting cells from oxidative stress in AKI [8,9]. (LLC-PK1) in vitro by means of mitochondria-dependent and - independent pathways [3], partly through the activation of

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Figure 1. Blood urea nitrogen (BUN) and serum creatinine (Cre) levels in ALA treated rats after cisplatin injection. Rats were divided into four subgroups: 1) a control (saline) group, 2) a cisplatin group, 3) an ALA–treated post cisplatin-injection group, 4) an ALA–treated pre & post cisplatin-injection group (n = 8 for each group). Serum creatinine (A) and blood urea nitrogen (B), and body weight (C) were measured at the indicated times. Data are mean 6 SEM of 8 rats per group. Statistically significant differences (*p,0.05 v.s. CDDP, #p,0.05 v.s. control) are indicated. doi:10.1371/journal.pone.0080850.g001

Changes in mitochondrial structure and membrane potential were nephrotoxicity without compromising the anticancer efficacy of reported in the proximal tubules during AKI [8,9]. cisplatin. 5-Aminolevulinic acid (ALA) is the naturally occurring metabolic precursor of an endogenously synthesized photosensitizer, Materials and Methods protoporphyrin IX (PpIX) [10–12]. ALA is widely distributed in animal cells, and it is a precursor of tetrapyrole compounds such as Induction of cisplatin-induced AKI heme, which is fundamentally important in aerobic energy Male Sprague-Dawley rats (Saitama Experimental Animal metabolism [13]. Here, we explored the relevance of ALA in Supply, Saitama, Japan) weighing 150–200 g were anesthetized protecting renal tubular cells in cisplatin-treated rats through the by intraperitoneal injection with sodium pentobarbital (30 mg/kg). attenuation of mitochondrial enzymes and the apoptotic pathway. Cisplatin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in Thus, ALA has the potential to prevent cisplatin nephrotoxicity saline at a concentration of 1 mg/mL. The rats were given a single without compromising the anticancer efficacy of cisplatin. This intraperitoneal injection of either a vehicle (saline) or cisplatin study was conducted to determine whether ALA affects the course (8 mg/kg body weight). 5-ALA 10 mg/kg + Fe (sodium ferrous of cisplatin-induced AKI. To achieve this, we examined differ- citrate, 15.7 mg/kg) dissolved in drinking water were administered ences in the renal function, histology, changes of mitochondrial to rats. 5-ALA (COSMO BIO co., ltd. Tokyo, Japan), and sodium enzymes, and tubular cell apoptosis in cisplatin-induced AKI. Our ferrous citrate (kindly provided by SBI Pharmaceuticals Co., Ltd., data demonstrated that ALA has the potential to prevent cisplatin Tokyo, Japan) were prepared. 5-ALA and Fe were purchased form

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Figure 2. Renal histopathology and scores for characteristic histological signs of renal injury in ALA treated rats with cisplatin- induced AKI. A, B) Kidneys were removed 5 days after injection with cisplatin (8 mg/kg) or control. The cisplatin-treated rats exhibited acute structural damage characterized by tubular necrosis, swelling and tubular dilation, extensive epithelial vacuolization, and hyaline casts in renal tubules. C, D) Kidneys of ALA treatment (both post and pre & post) show a very slight loss of tubular epithelial cells and very low levels of intratubular debris and cast formation (Magnification, X50 upper, X200 lower figures) E) ALA reduced the renal injury score in cisplatin-treated rats. The semiquantitative histological injury score was significantly higher in cisplatin-treated rats than in controls. Data are the mean SEM of 6 rats per group. Statistically significant differences (* p,0.05) are indicated. doi:10.1371/journal.pone.0080850.g002

Sigma-Aldrich (St. Louis, MO, USA). The animals were divided Effects of ALA on the antitumorigenic effects of cisplatin into 4 subgroups: (1) a control (saline) group, (2) a cisplatin group, Male F344/NJcl-rnu/rnu rats (immunodeficiency rats) (Saitama (3) an ALA-treated post-cisplatin-injection group (post), and (4) an Experimental Animal Supply) weighing 150–200 g were anesthe- ALA-treated pre- and post-cisplatin-injection group (pre & post) tized by intraperitoneal injection with sodium pentobarbital (n = 8 for each group). The blood was obtained via tail vein at 1, 3, (30 mg/kg). 253J-BV (a bladder carcinoma cell line) cells 5, 7, and 9 days after cisplatin injection. The rats were killed at 5 (26107) originally purchased from American Type Culture and 9 days after surgery (Figure S1). The left kidney was rapidly Collection (Manassas, VA, USA) were subcutaneously injected removed and processed for histological evaluation, protein into the skin of the back. The rats were given a single extraction, and RNA extraction at day 5 and 9 as previously intraperitoneal injection of either a vehicle (saline) or cisplatin described [14,15]. All animal protocols were approved by the (8 mg/kg body weight). 5-ALA 10 mg/kg + Fe (sodium ferrous Institutional Animal Care and Use Committee at the University of citrate, 15.7 mg/kg) dissolved in drinking water were administered Kochi (#20–027), and experiments were conducted in accordance to rats. The animals were divided into 4 subgroups: (1) a control with institutional guidelines. All surgery was performed under (saline) group, (2) a cisplatin group, (3) an ALA-treated post- sodium pentobarbital anesthesia, and all efforts were made to cisplatin-injection group (post), and (4) an ALA-treated pre- and minimize suffering. post-cisplatin-injection group (pre & post) (n = 5 for each group). The volume of the carcinoma was measured at 1, 3, 5, 7, and 9 days after surgery.

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Figure 3. Tubular cell apoptosis and levels of cleaved caspase3 in renal tissues of ALA treated rats with cisplatin-inducedAKI. Kidneys were removed 5 days after an injection with cisplatin (8 mg/kg). A, B) Kidneys of cisplatin-treated rats exhibited an elevated number of TUNEL-positive renal tubular cells. (Magnification, X100). C, D) Kidneys of ALA treatment (both post and pre & post) show a very few number of TUNEL-positive renal tubular cells. (Magnification, X100) E) The number of TUNEL positive tubular cells was significantly low in ALA (both post and pre & post) treated rat kidneys in cisplatin-induced AKI. (F) Western blot analysis of cleaved caspase3 were performed in each group rats. (G) Quantitative densitometry was performed for cleaved caspase 3 blots. Data are the mean 6 SEM of 6 rats per group. Statistically significant differences (*p,0.05) are indicated. doi:10.1371/journal.pone.0080850.g003

Cell culture then rapidly excised, frozen in liquid nitrogen, and homogenized NRK-52E cells (renal tubular cells from adult rats), originally in SDS sample buffer, as described previously [16]. For purchased from American Type Culture Collection, were grown immunohistochemical studies, kidneys were fixed in formalin in Dulbecco’s modified Eagle’s medium (Gibco, Rockville, MD, overnight, dehydrated, and embedded in paraffin. Thin sections USA) supplemented with 50 IU/mL penicillin and 10% heat- were cut and subjected to periodic acid-Schiff staining, as inactivated fetal calf serum (Gibco) [15]. For the cisplatin described previously. Tubular injury was assessed by using a experiments, cisplatin (20 mM) was added to the NRK-52E cells semiquantitative scale [6]. Histological changes due to tubular for 24 h. For the ALA + cisplatin experiments, 200 mM 5-ALA, necrosis were quantitated by determining the percentage of 100 mM sodium ferrous citrate, and 20 mM cisplatin were added tubules with evident cell necrosis, loss of brush border, cast to the NRK-52E cells for 24 h. All other chemicals were formation, and tubule dilatation, as follows: 0 = none, 1 = #10%, purchased from Funakoshi (Tokyo, Japan). 2 = 11%–25%, 3 = 26%–45%, 4 = 46%–75%, and 5 = #76%. At least 5–10 fields (6200) were reviewed for each slide. The Isolation and histological examination of kidney tissue Apoptosis TUNEL Kit II (MBL, Tokyo, Japan) was used for the Rats were anesthetized with pentobarbital at the indicated times staining of terminal deoxynucleotidyl transferase-mediated dUTP after cisplatin administration. The kidneys were perfused in situ nick end labeling (TUNEL)-positive cells, as previously described with sterile phosphate-buffered saline (PBS) and the left kidney was [6].

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Measurements of heme We measured heme to evaluate the metabolic product of ALA using assay kit (BioChain Institute, Newark, CA). Heme Assay Kit is based on an improved aqueous alkaline solution method, in which the heme is converted into a uniform colored form. The intensity of color, measured at 400 nm, is directly proportional to the heme concentration in the sample. We measured the renal tissue extract in the in vivo experiments, and the cell extracts in the in vivo experiments. The protein extracts of the total renal tissue or NRK-52E cells (100 mg samples) were prepared following the same method used in the immunoblot analysis.

Real-time quantitative polymerase chain reaction Reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA extracted from kidneys was carried out as previously described [16]. In brief, total RNA was isolated from renal tissues by using TRI Reagent (Life Technologies, Gaithers- burg, MD, USA). Samples of total RNA (1 mg) were reverse transcribed, and real-time qPCR was performed to quantify changes in ATP5a, COX-IV, PGC-1a, and UCP2 gene expression by using the ABI LightCycler real-time PCR system (ABI, Los Angeles, CA, USA). RT-PCR of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a positive control. A 3-step PCR was performed for 35 cycles. The samples were denatured at 94uC for 30 s, annealed at 58uC for 30 s, and extended at 72uC for 30 s. The primers were obtained from ABI.

Mitochondrial morphology obtained by using laser confocal immunofluorescence microscopy NRK-52E cells were stained with MitoTracker mitochondrion- selective probes (Invitrogen, Rockville, MD, USA). For confocal Figure 4. Western blot analyses of protein expression and RT- microscopy, NRK-52E cells were then fixed with 2% paraformal- PCR analysis of mitochondria-related gene expression in ALA dehyde in PBS for 1 h and processed for imaging as described treated cisplatin-induced AKI rats. (A) Aliquots of 50 mg of protein previously, and examined under a confocal laser microscope (Carl from renal tissue extracts were separated by SDS-PAGE and transferred Zeiss Japan, Tokyo, Japan) [18]. Fragmented mitochondria were to membranes. Western blots analyses were performed for ATP5a, complex (COX)-IV, PGC-1a, UCP2 in cisplatin-treated, control, and condensed and punctate, whereas normal mitochondria showed a cisplatin + ALA (both post and pre & post) treated rats. Actin served as a threadlike or rounded structure. Cells with mitochondrial loading control. (B) Quantitative densitometry was performed for fragmentation were defined as those containing a majority ATP5a,complex(COX)-IV,PGC-1a,andUCP2westernblots.(C) (.70%) of fragmented mitochondria, and they were counted to Quantitative analysis of mRNA was performed using RT-PCR for ATP5a, determine the percentage in 50 cells/sample. complex (COX)-IV, PGC-1a, and UCP2. GAPDH served as a loading control. Bars represent the mean 6 SEM, n = 6. *p,0.05 v.s. CDDP, #p,0.05 v.s. control by ANOVA. Statistics doi:10.1371/journal.pone.0080850.g004 Results are presented as mean 6 SEM. Differences between the groups were tested by 2-way analysis of variance (ANOVA) followed by Scheffe’s test for multiple comparisons. Two groups were compared by using unpaired t-tests. A p value of ,0.05 was Western blot analysis considered statistically significant. Protein extracts of the total renal tissue or NRK-52E cells (50 mg samples) were prepared and denatured by heating at 100uC Results for 5 min in SDS sample buffer as described previously [17]. The proteins were separated on 7.5% or 10%–20% polyacrylamide 5-Aminolevulinic acid protects from cisplatin-induced gels and transferred to nitrocellulose membranes. The membranes renal injury were blocked for 1 h with 5% (wt/vol) fat-free milk in PBS and Cisplatin increased the serum blood urea nitrogen and probed with the appropriate primary antibodies (anti-ATP5a, creatinine levels in comparison with the controls at days 5–9. anti-complex [COX]-IV, anti-PGC-1a, anti-UCP2, anti-nitrotyr- ALA treatments (both post and pre & post) significantly prevented osine [anti-NT], anti-procaspase-3, or anti-actin [Santa Cruz these changes in cisplatin-treated animals (Figure 1A,B). Body Biochemicals Inc., Santa Cruz, CA, USA]). The primary weight was not significantly different in rats before the beginning antibodies were detected with horseradish peroxidase (HRP)- of the treatment. Cisplatin reduced body weight gain in the test conjugated rabbit anti-goat IgG or HRP-donkey anti-rabbit IgG, animals. ALA treatments (both post and pre & post) significantly and visualized by using the Amersham ECL system (Amersham prevented, but did not normalize, cisplatin-induced weight loss Corp., Arlington Heights, IL, USA). (Figure 1C). The toxic effect of cisplatin was also confirmed by the detection of morphologic abnormalities in kidney slices. The histology results for the control rats were normal (Figure 2A). The

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Figure 5. Protective effects of ALA to cisplatin-induced oxidative stress and induction of Heme oxygenase (HO)-1 expression in vivo. (A, D) Aliquots of 50 mg of protein from renal tissue extracts were separated by SDS-PAGE and transferred to membranes. Western blots analyses were performed for HO-1 (A), nitrotyrosine (NT) (D) in cisplatin-treated, control, and cisplatin + ALA (both post and pre & post) treated rats. Actin served as a loading control. (B) Quantitative densitometry was performed for HO-1 western blots. (C) Quantitative analysis of mRNA was performed using RT-PCR for HO-1. GAPDH served as a loading control (E) Quantitative densitometry was performed for NT western blots. Bars represent the mean 6 SEM, n = 6. *P,0.05 by ANOVA. doi:10.1371/journal.pone.0080850.g005 cisplatin group exhibited acute structural damage characterized by ALA administration (both post and pre & post) on quantitative tubular necrosis, swelling and tubular dilation, extensive epithelial analysis (Figure 3E). Cleaved caspase-3 levels were high in the vacuolization, and hyaline casts in renal tubules (Figure 2B–D). renal tissue of rats treated with cisplatin. ALA treatments (both ALA reduced these tubular damages in cisplatin-treated rats post and pre & post) significantly lowered the elevated caspase-3 (Figure 2C,D). The semiquantitative histological injury score was levels in cisplatin-injected rat kidneys, as assessed by western significantly higher in cisplatin-treated rats than in controls blotting of the renal tissue and densitometric analysis (Figure 3F,G). (Figure 2E). 5-Aminolevulinic acid ameliorates cisplatin-induced 5-Aminolevulinic acid reduces cisplatin-induced reduction of mRNA and protein expression of apoptosis mitochondria-related genes in vivo Apoptosis in the kidney was assessed by using the TUNEL We next examined whether ALA protects the mitochondrial assay. Cisplatin increased the number of apoptotic nuclei enzymes from cisplatin injury. Because previous studies demon- compared with the control group (Figure 3A,B). ALA treatments strated that cisplatin caused a marked decrease in the expression of (both post and pre & post) significantly decreased the number of several mitochondrial enzymes, we examined typical enzymes TUNEL-positive cells (Figure 3C,D). The number of TUNEL- (ATP5a, COX-IV, PGC-1a, and UCP2) in cisplatin-treated, positive cells were increased by cisplatin treatment and reduced by control, and cisplatin + ALA–treated rats. As shown in Figure 4A,

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cisplatin treatment in vivo (Figure 5A–C). ALA treatments (both post and pre & post) further increased HO-1 expression (Figure 5A–C). Oxidative stress is a major factor causing renal injury in response to cisplatin. We examined the level of oxidative stress by using a typical marker, NT. On immunoblotting, NT was highly expressed in the cisplatin-treated kidney compared with the control (Figure 5D,E). However, treatments with ALA (both post and pre & post) reduced these signals (Figure 5D,E). These data suggest that cisplatin induced oxidative stress in damaged tubules, whereas such stress was significantly blocked by the ALA treatment.

5-Aminolevulinic acid and Fe protect against cisplatin- induced reduction of mRNA and protein expression of mitochondria-related genes in NRK-52E cells We next examined whether ALA and Fe protect the mitochondrial enzymes from cisplatin injury in vitro. We examined the typical enzymes (ATP5a, COX-IV, PGC-1a, and UCP2) in the control, cisplatin-, cisplatin + ALA-, and cisplatin + ALA + Fe– treated NRK-52E cells. As shown in Figure 6A,B, cisplatin induced a significant loss of ATP5a, COX-IV, PGC-1a, and UCP2 protein expression. ALA-, and ALA + Fe–treatment completely recovered the cisplatin-induced decreases in the expression of these proteins. Furthermore, to examine the changes in the mRNA expression of ATP5a, COX-IV, PGC-1a, and UCP2 after cisplatin treatment, we conducted RT-PCR analysis of mRNA in NRK-42E cells. The mRNA level of these enzymes was dramatically decreased by cisplatin, and these reductions were ameliorated by addition with ALA and, more effectively, ALA + Fe (Figure 6C).

Figure 6. Western blot analyses of protein expression and RT- ALA and Fe protect against cisplatin-induced oxidative PCR analysis of mitochondria-related gene expression in ALA + Fe treated cisplatin-induced renal tubular injury. (A) Aliquots of stress and induce HO-1 expression in NRK-52E cells 50 mg of protein extracts from NRK-52E cells were separated by SDS- We next examined the HO-1 expression in the presence of PAGE and transferred to membranes. Western blots analyses were cisplatin, ALA, and Fe. HO-1 mRNA and protein expression was performed for ATP5a, complex (COX)-IV, PGC-1a, UCP2 in control, induced by cisplatin treatment in NRK-52E cells. ALA treatment cisplatin, cisplatin + ALA, and cisplatin + ALA + Fe treated NRK-52E cells. additionally increased HO-1 expression. ALA + Fe treatment Actin served as a loading control. (B) Quantitative densitometry was performed for ATP5a, complex (COX)-IV, PGC-1a, and UCP2 blots. (C) significantly upregulated HO-1 expression in the presence of Quantitative analysis of mRNA was performed using RT-PCR for ATP5a, cisplatin (Figure 7A–C). Oxidative stress is a major factor causing complex (COX)-IV, PGC-1a, and UCP2. Bars represent the mean 6 SEM, renal injury in response to cisplatin. On immunoblotting, NT was n = 6. *p,0.05 v.s. CDDP, #p,0.05 v.s. control by ANOVA. highly induced by cisplatin treatment compared with the control. doi:10.1371/journal.pone.0080850.g006 Treatment with ALA and Fe significantly reduced these signals (Figure 7D,E). cisplatin induced a significant loss of ATP5a, COX-IV, PGC-1a, and UCP2 protein expression. ALA treatment (both post and pre & ALA and Fe prevent cisplatin-induced damage of post) recovered the cisplatin-induced decreases of ATP5a, COX- mitochondrial structure and apoptosis in NRK-52E cells a IV, PGC-1 , and UCP2 protein expression. Quantitative analysis We examined the effects of ALA and Fe on mitochondrial a revealed that cisplatin induced decreases of ATP5 , COX-IV, structure by using a laser-scanning confocal microscope. The a PGC-1 , and UCP2 protein expression, and these reductions were typical reticulotubular appearance of mitochondria in healthy ameliorated by ALA treatments (both pre and pre & post) NRK-52E cells (Figure 8A) had disintegrated into condensed (Figure 4B). Furthermore, to examine the changes in the expression rounded organelles in response to cisplatin at 12 h (arrows in of these enzymes during cisplatin-induced AKI, we conducted an Figure 8B). However, ALA and Fe treatment prevented these RT-PCR analysis of rat renal tissue mRNA. The mRNA levels of structural changes in mitochondria (Figure 8D,E). ALA-only a a ATP5 , COX-IV, PGC-1 , and UCP2 were dramatically treatment partially prevented these structural changes in mito- decreased by cisplatin, and these reductions were ameliorated by chondria (Figure 8C,E). We also examined the effects of ALA and ALA treatments (both pre and pre & post) (Figure 4C). Fe in cisplatin-induced apoptosis in NRK-53E cells. We used TUNEL staining to evaluate apoptosis in NRK-52E cells, and 5-Aminolevulinic acid protects against cisplatin-induced found that cisplatin-induced apoptosis was significantly reduced by oxidative stress and induces heme oxygenase-1 ALA and ALA + Fe (Figure 9A,B). These data are in accordance expression in vivo with the results of western blot analysis of cleaved caspase-3 We next examined the renal HO-1 expression in the 4 groups of (Figure 9C,D). rats. HO-1 mRNA and protein expression was induced by

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Figure 7. Protective effects of ALA + Fe to cisplatin-induced oxidative stress and induction of Heme oxygenase (HO)-1 expression in NRK-52E cells. (A, D) Aliquots of 50 mg of protein extracts from NRK-52E cells were separated by SDS-PAGE and transferred to membranes. Western blots analyses were performed for HO-1 (A), nitrotyrosine (NT) (D) in control, cisplatin, cisplatin + ALA, and cisplatin + ALA + Fe treated NRK-52E cells. Actin served as a loading control. (B) Quantitative densitometry was performed for HO-1 blots. (C) Quantitative analysis of mRNA was performed using RT-PCR for HO-1. (E) Quantitative densitometry was performed for NT blots. Bars represent the mean 6 SEM, n = 6. *P,0.05 by ANOVA. doi:10.1371/journal.pone.0080850.g007

Measurement of heme in ALA-treated, cisplatin-induced increased the heme concentration. These data demonstrated that AKI rats and NRK-52E cells exposed to cisplatin and ALA + heme is up-regulated by ALA treatment both in vitro and in vivo. Fe We used a heme assay kit to evaluate the metabolic products of Effects of 5-Aminolevulinic acid on the antitumorigenic ALA. We examined the heme concentration in the renal tissues in effects of cisplatin in vivo the cisplatin-treated, control, and cisplatin + ALA-treated rats. As To evaluate if the ALA reduction of cisplatin nephrotoxicity was shown in Figure 10A, cisplatin did not significantly change the specific for the kidney, we looked for potential reductions in the heme concentration. ALA treatment (both post and pre + post) chemotherapeutic efficacy of cisplatin in bladder carcinoma cells. significantly increased the heme concentration. We then examined We evaluated the size of the renal carcinoma transplanted into the whether ALA and Fe increased heme concentration in vitro. We rat skin, as shown in Figure 11, and found that cisplatin reduced measured the heme concentrations in the control, cisplatin-, the size. ALA (post and pre & post) did not change the anticancer cisplatin + ALA-, and cisplatin + ALA + Fe-treated NRK-52E effects of cisplatin. cells. As shown in Figure 10, cisplatin did not change the heme concentration. ALA as well as ALA + Fe treatment significantly

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tubule cells in the outer medulla of the kidney [20,21]. The cellular events in cisplatin-mediated nephrotoxicity, including decreased protein synthesis, membrane peroxidation, mitochondrial dysfunction, and DNA injury, are a consequence of free radical generation and the body’s inability to scavenge such molecules [22,23]. Consistent with previous studies, we found that cisplatin- induced renal dysfunction and morphological changes were associated with mitochondrial injury in the rat kidney [9]. These findings were also confirmed by using NRK-52E cells, in which we documented that cisplatin caused mitochondrial fragmentation and decrease of mitochondrial enzymes. In animal cells, ALA is formed from glycine and succinyl CoA by ALA synthase in mitochondria. COX and cytochrome c are hemoproteins. In this study, we demonstrated that COX-IV expression was increased by ALA administration in vivo and in vitro. This finding is in accordance with previous report with liver lysates [24]. ALA is the precursor of protoporphyrin, and heme is produced by the insertion of iron. Therefore, ALA administration can result in heme production in the rat kidney, and it is possible that COX-IV was increased by ALA administration. ALA administration upregulates HO-1 expression. HO-1 is a key enzyme for antioxidant response in renal tubular cells and therefore protects mitochondrial function and upregulates mitochondrial enzymes. We first demonstrated that typical mitochondrial enzymes, such as PGC-1a, UCP2, and ATP5a, were upregulated by ALA administration. Interestingly, ALA and Fe strongly induced these enzymes. This is the first study to demonstrate that HO-1 is upregulated by ALA and Fe in vivo and in vitro. HO-1 a microsomal enzyme involved in the degradation of heme, resulting in the generation of biliverdin, iron, and carbon monoxide. Recent attention has focused on the biological effects of product(s) of this enzymatic reaction that have important antioxidant, anti-inflammatory, and cytoprotective functions [25]. Induction of HO-1 occurs as an adaptive and beneficial response to a wide variety of oxidant Figure 8. ALA and Fe prevent cisplatin-induced damage of stimuli, including heme, hydrogen peroxide, cytokines, growth mitochondrial structure in NRK-52E cells. (A–D) Confocal micros- factors, heavy metals, nitric oxide, and oxidized LDL [25]. HO-1, copy demonstrates mitochondria structure in NRK-52E cells transfected with mitochondria-targeted red fluorescent protein (mitoDsRed) the enzyme that is responsible for heme degradation, is vectors. NRK-52E cells were treated with in control condition, cisplatin, upregulated in the proximal tubule cells in response to oxidant cisplatin + ALA, and cisplatin + ALA + Fe. Typical condensed rounded stress [26], and once induced, it confers dramatic cytoprotective organelles in response to cisplatin were marked by arrows. (E) and anti-inflammatory effects [25,27]. The mechanisms of HO-1 Quantitative analysis of the typical reticulotubular appearance of regulation are reported by several pathways: one is hypoxia and mitochondria in healthy NRK-52E cells and multiple rounded organelles are performed. ALA and Fe treatment prevented these structural inflammatory signals, including IL-1 and TNFa, and second are changes in mitochondria. ALA only treatment partially prevented these the nuclear factor E2-related factor-2 (Nrf2) and heme levels. HO- structural changes in mitochondria. Bars represent the mean 6 SEM, 1 gene regulation is reported to involve Kelch-like enoyl-CoA n = 6. *P,0.05 by ANOVA. hydratase (ECH)-associated protein 1 (Keap1) regulation through doi:10.1371/journal.pone.0080850.g008 antioxidant response elements (ARE), Nrf2, and their binding in the cytosol [28]. Recently, investigators identified a heme- dependent degradation system involving iron regulatory protein 2 (IRP2) as a sensor of iron metabolism. IRP2 upregulates Discussion intracellular free iron and modulates intracellular iron stores, and increased iron efflux has been suggested as a mechanism for the In this study, we demonstrated that the protective role of ALA cytoprotective effects of HO-1 expression [29]. We clearly in cisplatin-induced AKI is through the protection of mitochon- demonstrated that cisplatin itself upregulates HO-1 expression; drial viability, and ALA prevents tubular apoptosis. In addition, these findings were in accordance with those of the previous ALA has no significant effects on the anticancer efficiency of reports [30,31]. Furthermore, we demonstrated that ALA induced cisplatin in rats. Thus, ALA has the potential to prevent cisplatin HO-1 and ALA + Fe additively upregulated HO-1 mRNA and nephrotoxicity without compromising the anticancer efficacy of protein expression in NRK-52E cells. The additional induction of cisplatin. Cisplatin is a chemotherapeutic agent that is used in the HO-1 by Fe may confer cytoprotective and antioxidant responses treatment of a variety of solid-organ cancers, including those of the in renal tubular cells and protect mitochondrial function and head, neck, testis, ovary, and breast [19]. Unfortunately, in enzymes such as PGC-1a, ATP5a, and UCP2. However, further addition to causing bone marrow suppression, ototoxicity, and studies are needed to completely clarify the mechanisms of heme anaphylaxis, around 30% of patients receiving cisplatin develop regulation and their associated metabolic pathways concerning AKI owing to its preferential accumulation within the proximal mitochondrial function.

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Figure 9. TUNEL assay and cleaved caspase 3 to evaluate apoptosis in NRK-52E cells exposed to cisplatin and ALA + Fe. (A) TUNEL assay to evaluate apoptosis (green) in NRK-52E cells exposed to cisplatin or control. Nuclei were stained with propidium iodide (PI) (red). TUNEL assay to evaluate apoptosis (green) in NRK-52E cells exposed to cisplatin + ALA or cisplatin + ALA + Fe. (B)The induction of the number of apoptotic cells by cisplatin was reduced by ALA and ALA + Fe using quantitative analysis. Data are presented as mean 6 SEM, n = 6. *P,0.05 vs. pcDNA-transfected cells. (C) Western blot analysis of cleaved caspase3 were performed following incubation with in control condition, cisplatin, cisplatin + ALA, and cisplatin + ALA + Fe. (D) Quantitative densitometry was performed for cleaved caspase 3 blots. Data are presented as mean 6 SEM, n = 6. *P,0.05 by ANOVA. doi:10.1371/journal.pone.0080850.g009

No previous studies before ours have demonstrated the suggested that ALA induces apoptosis through the accumulation of protective effects of ALA against cisplatin-induced apoptosis in PpIX. ALA-mediated accumulation of PpIX causes photosensiti- vivo and in vitro. Cisplatin induces apoptosis of renal proximal zation of cancer cells and is used in the treatment of hepatocellular tubule cells (LLC-PK1) in vitro through mitochondria-dependent carcinoma, oral cancer, and bladder carcinoma [13,33]. In several and -independent pathways [3], partly through the activation of cancer cell lines, ABCG2 transporter is highly expressed and PpIX caspase-3 and oxidative stress [4,5]. Several studies suggest that is accumulated in the cytosol and causes cytotoxic damage and caspase inhibitors or knockout of apoptosis-related genes attenuate apoptosis [10]. The mechanisms of the differential effects of ALA ischemia-induced AKI in rats [32]. In our experiments, cisplatin in the apoptotic pathway in normal renal tubular cells and administration induced apoptosis in vivo and vitro, as confirmed carcinoma cells are not well known and need to be studied in by TUNEL staining and cleaved caspase-3 level. Our data clearly future. demonstrated that ALA inhibited cisplatin-induced apoptosis in Cisplatin is one of the most effective and potent anticancer vivo and vitro, which was evaluated by examining the results of drugs in the treatment of epithelial malignancies [1]. Considering TUNEL staining and cleaved caspase-3 levels. Furthermore, ALA the clinical use of ALA in preventing cisplatin-induced nephro- + Fe additionally reduced cisplatin-induced apoptosis in NRK- toxicity, it should be checked whether ALA interferes with the 52E cells. The plausible mechanisms of the ALA + Fe effects on anticancer effects of cisplatin. Thus, we examined the effects of antiapoptosis are antioxidative effects and protective effects toward ALA on the size of the renal carcinoma transplanted into rat skin. mitochondria in renal tubular cells. In cancer cells, several studies As shown in Figure 10, cisplatin reduced the size of the

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Figure 10. Measurement of heme in the ALA-treated cisplatin-induced AKI rats and NRK-52E cells exposed to cisplatin and ALA + Fe. (A) Aliquots (100 mg) of protein from renal tissue extracts were used for the heme assay in cisplatin-treated, control, and cisplatin + ALA (both post and pre + post)- treated rats. (B) Aliquots (10 mg) of protein extracts from NRK-52E cells were used for the heme assay in the control as well as the cisplatin-, cisplatin + ALA-, and cisplatin + ALA + Fe-treated NRK-52E cells. Data are the mean 6 SEM of six experiments per group. *P,0.05 v.s. control or CDDP, n.s. is not significant by ANOVA. doi:10.1371/journal.pone.0080850.g010 transplanted renal carcinoma. ALA (post and pre & post) did not to gain insight into the effects of ALA on the anticancer effects of change the anticancer effects of cisplatin. At least in our cisplatin before clinical use. experimental condition, ALA did not interfere with the anti- In summary, our study has produced 2 novel findings. First, the tumorigenic effects of cisplatin in vivo. Further research is needed protective role of ALA in cisplatin-induced AKI is through the protection of mitochondrial viability, induction of HO-1, and prevention of tubular apoptosis. Second, ALA has no significant effects on the anticancer efficiency of cisplatin in rats and prevents tubular apoptosis. Further studies are necessary to gain a more precise understanding of the molecular mechanisms by which ALA protects renal cells against cisplatin-induced nephrotoxicity.

Supporting Information Figure S1 Experimental designs for in vivo study. The rats were given a single intraperitoneal injection of either a vehicle (saline) or cisplatin (8 mg/kg body weight). 5-Aminolevulinic acid (ALA) 10 mg/kg + Fe (sodium ferrous citrate 15.7 mg/kg) were dissolved in drinking water (10 ml/kg) were administered. Rats were divided into four subgroups: 1) a control (saline) group, 2) a cisplatin group, 3) an ALA–treated post (0–9 days after CDDP injection) cisplatin-injection group, 4) an ALA–treated pre(5 days before CDDP injection) & post cisplatin-injection group (n = 8 for each group). Blood samples were obtained for measurement of blood urea nitrogen and serum creatinine. at 1, 3. 5, 7, and 9 days after CDDP injection. Rats were sacrificed at day 5 and 9, and renal tissue are obtained. (TIF) Figure 11. Effects of ALA on anti-tumorinogenic effects of cisplatin in vivo. We evaluated the size of transplantated bladder Acknowledgments carcinoma to the F344/NJcl-rnu/rnu rats skin. Rats were divided into four subgroups: 1) a control (saline) group, 2) a cisplatin group, 3) an We thank Ms. Chiaki Kawada, Ms. Reiko Matsumoto, Ms. Sekie Saito, ALA–treated post cisplatin-injection group, 4) an ALA–treated pre & Ms. Akiko Takano, Mr. Yasushi Okada, Ms. Yoko Akimaru, and Mr. post cisplatin-injection group (n = 5 for each group), The diameter of Tomomi Anma for technical assistances. We thank Dr. Tohru Tanaka, Dr. the carcinoma were measured at 1, 3. 5, 7, and 9 days after surgery Motowo Nakajima, Dr. Atsuko Kamiya, Dr. Kyoko Tsuchiya (SBI (n = 5/group). Cisplatin reduced size of transplantated renal carcinoma Pharmaceuticals Co., Ltd., Tokyo, Japan) for kindly providing sodium compared with control group. ALA (post and pre & post) did not ferrous citrate and for helpful discussions. change the anti cancer effects of cisplatin. Data are the mean 6 SEM of 6 experiments per group. *P,0.05 v.s. control by ANOVA. doi:10.1371/journal.pone.0080850.g011

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Author Contributions Analyzed the data: Y. Terada Keiji Inoue TM MI KH YS TK KT HF SF MT. Contributed reagents/materials/analysis tools: Y. Terada Conceived and designed the experiments: Y. Terada Keiji Inoue MT, Keiji Inoue MT TS. Wrote the paper: Y. Terada Keiji Inoue TS. TS. Performed the experiments: Y. Terada Keiji Inoue TM MI KH YS KO Kosuke Inoue Y. Taniguchi TH TK KT HF SF MT.

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